Thermodynamic model for micelle formation by phosphatidylcholines containing short-chain fatty acids. Correlations with physical-chemical data and the effects of concentration on the activity of phospholipase A2

Allgyer, Thomas T.; Wells, Michael A.

doi:10.1021/bi00587a014

Cited by 19 publications

(12 citation statements)

References 34 publications

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“…A comparison of the amount of hemoglobin released by pure SLUV mixture at 25 °C to the amount released by diheptanoyl-PC suggests ~0.1 mM of the short-chain lecithin may be free (i.e., not in the bilayer). This concentration of monomers would yield a specific activity of <50 µ min"1 mg"1 for phospholipase A2 (Pieterson et al, 1974; Allgyer & Wells, 1979). The high activity at 45 °C is on a SLUV preparation with little, if any, free diheptanoyl-PC (none above background is detected with the hemolysis assay).…”

Section: Resultsmentioning

confidence: 99%

Enzymic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state

Nv²,

Mf³

1987

Biochemistry

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Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.

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Section: Resultsmentioning

confidence: 99%

Enzymic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state

Nv²,

Mf³

1987

Biochemistry

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“…These results can be taken to support the 'substrate theory' of PLA2 activation at the CMC [4][5][6][7][8][9][10][11] as they reflect changes in the conformation of phospholipids due to incorporation of the lipid into a mixed micelle. The observed changes in the lipid conformation influencing the action of PLA2 also seem to be in line with our earlier model developed on the basis of results from a distinctly different experimental approach [20,21].…”

Section: Discussionmentioning

confidence: 60%

“…However, the zymogen of pancreatic PLA2 is not activated at interfaces [3]. The most feasible explanations for the interfacial activation are: (i) the substrate theory which assumes substrate molecules to be in interfaces in a conformation different from that in monomolecular solutions and resulting in an enhanced PLA2 activity [4][5][6][7] or alternatively, assumes the dehydration of interfacial substrate to be responsible for the interfacial activation [8][9][10][11] and (ii) the enzyme theory which assumes a conformational change in the enzyme molecule upon adsorption to the interface [3,12,13]. These models are not mutually exclusive.…”

Section: Introductionmentioning

confidence: 99%

A model for the molecular mechanism of interfacial activation of phospholipase A₂ supporting the substrate theory

Thurén

1988

FEBS Letters

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Changes occurring in the activity of porcine pancreatic phospholipase A 2 upon formation of mixed micelles of sodium cholate and the fluorescent phosphocholines 1,2-di [6-(pyren-l-yl)butanoyl]-sn-glycero-3-phosphocholine or 1-[6-(pyren-1-yl)butanoyl]-2-[6-(pyren-1-yl)hexanoyl]-sn-glycero-3-phosphochofine were studied. A 2-fold enhancement was observed in the activity of phospholipase A~ towards both pyrene phospholipids upon exceeding the critical micellar concentration of the system. Changes in the pyrene excimer/monomer fluorescence emission intensity ratio coincide with the enhancement of phospholipase A 2 activity at the critical mieellar concentration. Due to the different effects of micellization on the alignment of the pyrene in the two fluorescent probes conformational changes could be assessed. A model describing possible conformations of these pyrene phospholipid molecules below and above the critical micellar concentration is presented and correlated with the interfacial activation of phospholipase A 2.

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“…In this scheme, the number 2.4 is pATm, the negative log of the Michaelis constant [from Allgyer & Wells (1979)], while the number for the vertical reaction is taken from Figure 1. In this scheme, L refers to the diC6PC substrate.…”

Section: Discussionmentioning

confidence: 99%

“…We have not yet found a micellar nucleation site on the Crotalus PLPA2, but only a few experiments have been performed outside the conditions of Table I so we currently have no information whether this enzyme might show the effect under other pH or substrate conditions. Possibly this enzyme may only influence the diC6PC substrate above the cmc (Allgyer & Wells, 1979;Johnson et al, 1981).…”

Section: Discussionmentioning

confidence: 99%

Self-association and active enzyme forms of Naja naja naja and Crotalus atrox phospholipase A2 studied by analytical ultracentrifugation

Bukowski¹,

Teller²

1986

Biochemistry

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The dimerization of phospholipase A2 (PLPA2) from Naja naja naja (Pakistani cobra) and Crotalus atrox (Western Diamondback rattlesnake) has been studied from pH 2.5 to 11 at 20 degrees C in 1 mM CaCl2, 0.21 M ionic strength. For the C. atrox enzyme, it was found necessary to use a combination of sedimentation equilibrium and fluorescence yield data to analyze the association. Sedimentation equilibrium in the analytical ultracentrifuge sufficed for the study of the N. naja PLPA2. In the region of enzymatic activity at pH 8, the dimerization association constants found were k2 = 2.8 X 10(6) L/mol and k2 = 6.9 X 10(4) L/mol for the C. atrox and N. naja enzymes, respectively. Analytical linked functions are presented which describe the data. Because the associations are linked to Ca2+ as well as the hydrogen ion, no attempt was made to interpret the ionization of residues in terms of the molecular structure. Active-enzyme sedimentation velocity experiments have been used to study the relation between enzymatic activity and association for both the C. atrox and N. naja enzymes. The substrate 1,2-dibutyryl-sn-glycero-3-phosphocholine (diC4PC) did not dissociate the C. atrox PLPA2. The substrate 1,2-dihexanoyl-sn-glycero-3-phosphocholine (diC6PC) at 7.5 mM dissociated the C. atrox PLPA2 when monitored either as the active enzyme or as the Sr2+-inhibited enzyme. At low enzyme concentrations, 40 mM diC4PC had no effect on N. naja PLPA2 dimerization. However, the sedimentation coefficients observed at enzyme concentrations above 0.2 mg/mL in active-enzyme sedimentation velocity experiments were larger than the values predicted from the thermodynamic studies. Sedimentation coefficients observed for the N. naja PLPA2 acting on diC6PC were larger than those of the monomeric protein, which was the form layered on this substrate. The dissociation of the C. atrox PLPA2 effected by diC6PC was analyzed by the thermodynamics of association and the kinetic Michaelis constant. The analysis suffices to account for the observed sedimentation coefficient. The sedimentation behavior of the N. naja PLPA2 acting on diC6PC substrate was analyzed in terms of a protein-lipid complex. With this model, 68 +/- 16 phospholipid molecules per protein monomer were determined. It is proposed that this enzyme has two micelle nucleation sites per monomer. These putative sites promote micelle formation of the substrate on the enzyme below the critical micelle concentration of the lipid alone.

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Thermodynamic model for micelle formation by phosphatidylcholines containing short-chain fatty acids. Correlations with physical-chemical data and the effects of concentration on the activity of phospholipase A2

Cited by 19 publications

References 34 publications

Enzymic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state

Enzymic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state

A model for the molecular mechanism of interfacial activation of phospholipase A₂ supporting the substrate theory

Self-association and active enzyme forms of Naja naja naja and Crotalus atrox phospholipase A2 studied by analytical ultracentrifugation

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Thermodynamic model for micelle formation by phosphatidylcholines containing short-chain fatty acids. Correlations with physical-chemical data and the effects of concentration on the activity of phospholipase A2

Cited by 19 publications

References 34 publications

Enzymic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state

Enzymic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state

A model for the molecular mechanism of interfacial activation of phospholipase A2 supporting the substrate theory

Self-association and active enzyme forms of Naja naja naja and Crotalus atrox phospholipase A2 studied by analytical ultracentrifugation

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A model for the molecular mechanism of interfacial activation of phospholipase A₂ supporting the substrate theory